Lipid A (endotoxin) is the hydrophobic anchor of lipopolysaccharide (LPS) in the outer membrane of gram-negative bacteria (FIG. 1), and is an attractive antimicrobial target for three principal reasons. First, lipid A is essential for growth of E. coli and many other gram-negative pathogens (Raetz and Whitfield, 2002, Annu Rev Biochem 71: 635-700; Wyckoff et al., 1998, Trends Microbiol 6: 154-159). Second, decreased synthesis of lipid A can disrupt the integrity of the outer membrane, rendering bacteria more susceptible to other antibiotics. Finally, lipid A is one of the most potent immunostimulatory agents known, and is recognized by the TLR4 receptor in the mammalian innate immune system (Kaisho and Akira, 2002, Biochim Biophys Acta. 1589: 1-13). Endotoxic (septic) shock is one of the leading causes of mortality in intensive care units, responsible for over 100,000 deaths annually in North America (Kaisho and Akira, 2002). Thus, inhibition of lipid A synthesis: (i) directly kills pathogenic bacteria, (ii) makes them more susceptible to existing antibiotics, and (iii) simultaneously decreases levels of circulating endotoxin to prevent septic shock in infected patients. A specific inhibitor (L-573,655) of another enzyme in lipid A biosynthesis that is not ACP dependent (LpxC—catalyzes the deactylation of UDP-3-acyl-GlcNAc) has been identified and shows bacteriocidal activity against a broad variety of gram-negative pathogens, including E. coli and Pseudomonas aeruginosa (Wyckoff et al., 1998; Onishi, 1996, Science 274: 980-982).
All enzymes involved in E. coli lipid A biosynthesis have now been identified, and their structural genes have been cloned. The first step in lipid A biosynthesis is catalyzed by UDP-N-acetylglucosamine (UDP-GlcNAc) acyltransferase (LpxA), which transfers a β-hydroxy-fatty acyl group (typically 10-14 carbons in length, depending on the bacterial species) from ACP to the 3-OH glucosamine of UDP-GlcNAc (FIG. 1B). The x-ray structure of the E. coli (Raetz and Roderick, 1995, Science 270: 997-1000) and Helicobacter pylori (Lee and Suh, 2003, Proteins: Structure, Function and Genetics 53: 772-774) enzymes have been determined and are trimers of identical 30 kDa subunits. Chemical modification studies (Wyckoff and Raetz, 1999, J Biol Chem 274: 27047-27055) have indicated that the active site of LpxA is in a cleft shared by two adjacent subunits (FIG. 2). At one end of this cleft is an essential histidine residue (His-125) that promotes acyl transfer by general base catalysis (Wyckoff and Raetz, 1999), while the opposite end contains a glycine residue (Gly-173) that appears to act as a “hydrocarbon ruler” to determine fatty acid chain length specificity (Wyckoff et al., 1998, J Biol Chem 273: 32369-32372). The acidic acyl-ACP substrate may fit into an electropositive groove formed at the C-terminal contact regions between adjacent LpxA subunits (Lee and Suh, 2003). The Km values for UDP-GlcNAc and R-3-hydroxymyristoyl-ACP are 1 mM and 1 μM, respectively, and although myristoyl-ACP binds LpxA with similar affinity, it is not active as a substrate (Wyckoff and Raetz, 1999). LpxA is a potentially attractive drug target E. coli conditional lpxA mutants that exhibit <10% wild type LpxA activity are non-viable (Wyckoff et al., 1998). Moreover, even modest reduction (<30% decrease) of lipid A content in these mutants permits growth but increases sensitivity to erythromycin and rifampicin by >100-fold (Wyckoff et al., 1998).
ACP is also required for many other lipid products essential for bacterial growth and pathogenesis, including phospholipids (Rock and Jackowski, 1982, J Biol Chem 257: 10759-10765), acylated protein toxins such as hemolysin (Issartel et al., 1991, Nature 351, 759-761), lipoic acid (Jordan and Cronan, 1997, J Biol Chem 272: 17903-17906), polyketides (Shen et al., 1992, J Bacteriol 174: 3818-3821), and the acyl homoserine lactones involved in bacterial quorum sensing, ie. regulation of the timed release of bacterial toxins and biofilm formation (Parsek and Greenberg, 2000, Proc Natl Acad Sci 97: 8789-8793). Bacterial ACP is a small (70-90 amino acid) protein to which fatty acyl groups are attached as thioesters during fatty acid synthesis, and acyl-ACPs interact directly with at, least two dozen enzymes in a typical bacterial cell (FIG. 3). The flexible yet highly conserved tertiary structure of bacterial ACP is dominated by three parallel α-helices; helix II appears to be the principal region involved in enzyme binding (Parris et al., 2000, Structure Fold Des 8: 883-895; Zhang et al., 2001, J Biol Chem 276: 8231-8238) although other ACP residues are also involved (Flaman et al., 2001, J Biol Chem 276: 35934-35939). The conserved structural features of bacterial ACPs, together with the fundamental architectural differences with mammalian fatty acid synthases (where ACP exists as a discrete domain within a large multifunctional protein), make ACP/acyl-ACP binding a potential target for the development of broad specificity antimicrobials. Indeed, several natural or synthetic compounds have been identified that inhibit specific fatty acid synthase subunits. The broad-spectrum compound triclosan and the anti-tuberculosis drug isoniazid both inhibit enoyl-ACP reductases (FabI), while thiolactomycin and 3-decynoyl-NAC inhibit condensing enzymes (FabB) and dehydratase/isomerase (FabA), respectively (Heath et al., 2002, Appl Microbiol Biotechnol 58: 695-703). The present investigators have developed specialized methods to engineer, overexpress, and purify large amounts of recombinant holo-ACP from the bacterium Vibrio harveyi (Flaman et al., 2001), and have isolated a novel enzyme (V. harveyi acyl-ACP synthetase), providing the capacity to produce wild type or mutant ACPs and the specific acylated derivatives that are substrates for key essential bacterial processes (Shen et al., 1992, Anal Biochem 204: 34-39; Fice et al., 1993, J Bacteriol 175: 1865-1870).